Cardiac contractility depends on calcium sensitivity of the myofilaments and cytosolic free calcium concentration ([Ca(2+)](i)) during activation. During development, the cardiac troponin T isoform cTnT(1) is replaced by shorter cTnT isoforms, including cTnT(4), and changes occur in other myofibrillar proteins and in calcium regulation. We expressed rabbit recombinant (r)cTnT(1) and rcTnT(4) in Spodoptera frugiperda cells and determined their effect on calcium binding to TnC in solution and on the calcium sensitivity of myofilaments in skinned rabbit ventricular fibers in vitro. We measured [Ca(2+)](i) and L-type calcium current (I(Ca)) in ventricular myocytes from 3-wk-old and adult rabbits. The dissociation constant (K(d)) of Ca-Tn(cTnT1) in solution was smaller than that of Ca-Tn(cTnT4) (mean +/- SE: 0.52 +/- 0.08 mumol/L versus 0.83 +/- 0.09 mumol/L). The Ca(2+) sensitivity of force development was greater in fibers reconstituted with rcTnT(1) (pCa(50) 6.07 +/- 0.04) than those reconstituted with rcTnT(4) (pCa(50) 5.75 +/- 0.07). Systolic [Ca](i) was lower in 3-wk-old than adult cells (443 +/- 35 nmol/L versus 882 +/- 88 nmol/L) as was I(Ca) (5.8 +/- 0.9 pA/pF versus 14.2 +/- 1.6 pA/pF). The higher calcium sensitivity of Tn-Ca binding and of force development conferred by rcTnT(1) suggest that higher neonatal cTnT(1) expression may partially compensate for the lower systolic [Ca(2+)](i).